U.S. patent number 8,470,378 [Application Number 13/414,197] was granted by the patent office on 2013-06-25 for anti-influenza viral composition containing bark or stem extract of alnus japonica.
This patent grant is currently assigned to RNL Bio Co., Ltd. The grantee listed for this patent is Byeung Gie Kim, Hyuk Joon Kwon, Jeong Chan Ra, Seok Hyon You. Invention is credited to Byeung Gie Kim, Hyuk Joon Kwon, Jeong Chan Ra, Seok Hyon You.
United States Patent |
8,470,378 |
Ra , et al. |
June 25, 2013 |
Anti-influenza viral composition containing bark or stem extract of
Alnus japonica
Abstract
The present invention relates to an antiviral composition
comprising an Alnus japonica extract, more specifically, relates to
a method for preparing high activated anti-influenza viral
composition, which comprises an extract of the bark or stem of
Alnus japonica, and an anti-influenza viral composition comprising
the extract. An extract of the bark or stem of Alnus japonica
according to the present invention has low toxicity to normal
cells, while having an excellent antiviral effect even when
administered at low concentration and thus the composition
comprising the Alnus japonica extract can be used effectively in
preventing and treating influenza viral infection.
Inventors: |
Ra; Jeong Chan (Gyeonggi-do,
KR), Kwon; Hyuk Joon (Seoul, KR), Kim;
Byeung Gie (Gyeonggi-do, KR), You; Seok Hyon
(Seoul, KR) |
Applicant: |
Name |
City |
State |
Country |
Type |
Ra; Jeong Chan
Kwon; Hyuk Joon
Kim; Byeung Gie
You; Seok Hyon |
Gyeonggi-do
Seoul
Gyeonggi-do
Seoul |
N/A
N/A
N/A
N/A |
KR
KR
KR
KR |
|
|
Assignee: |
RNL Bio Co., Ltd (Seoul,
KR)
|
Family
ID: |
40755965 |
Appl.
No.: |
13/414,197 |
Filed: |
March 7, 2012 |
Prior Publication Data
|
|
|
|
Document
Identifier |
Publication Date |
|
US 20120164253 A1 |
Jun 28, 2012 |
|
Related U.S. Patent Documents
|
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
Issue Date |
|
|
12747289 |
|
|
|
|
|
PCT/KR2008/007172 |
Dec 4, 2008 |
|
|
|
|
Foreign Application Priority Data
|
|
|
|
|
Dec 11, 2007 [KR] |
|
|
10-2007-0127996 |
|
Current U.S.
Class: |
424/725 |
Current CPC
Class: |
A61P
31/16 (20180101); A61K 36/185 (20130101); A61P
31/12 (20180101) |
Current International
Class: |
A61K
36/00 (20060101) |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
|
|
|
|
|
|
|
08239307 |
|
Sep 1996 |
|
JP |
|
20030074500 |
|
Sep 2003 |
|
KR |
|
10-2004-0016578 |
|
Feb 2004 |
|
KR |
|
1020060023093 |
|
Mar 2006 |
|
KR |
|
620511 |
|
Sep 2006 |
|
KR |
|
100708593 |
|
Apr 2007 |
|
KR |
|
1020060026591 |
|
Apr 2007 |
|
KR |
|
100721703 |
|
May 2007 |
|
KR |
|
100769050 |
|
Oct 2007 |
|
KR |
|
00/40269 |
|
Jul 2000 |
|
WO |
|
2008/001976 |
|
Jan 2008 |
|
WO |
|
Other References
Balch, Prescription for Nutritional Healing, fourth edition,
section element of health, p. 98, copyrighted 2006. cited by
applicant .
Yu Young-Beob et al., Effects of triterpenoids and flavonoids
isolated from Alnus firma on HIV-1 viral enzymes, Archives of
Pharmacal Research, 2007, vol. 30, pp. 820-826. cited by applicant
.
Supplementary European Search Report and Opinion for EP 08 85 8731,
issued Jun. 5, 2011 by the European Patent Office. cited by
applicant.
|
Primary Examiner: Flood; Michele
Assistant Examiner: Winston; Randall
Attorney, Agent or Firm: Fuierer; Tristan A. Gerschutz;
Andrew D. Moore & Van Allen, PLLC
Parent Case Text
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a divisional application of and claims the
priority of U.S. patent application Ser. No. 12/747,289 filed on
Jun. 10, 2010 entitled "Anti-Influenza Viral Composition Containing
Bark or Stem Extract of Alnus Japonica" in the name of Jeong Chan
RA, et al., which claims priority of International Patent
Application NO. PCT/KR2008/007172 filed on Dec. 4, 2008, which
claims priority of Korean patent application Ser. No.
10/2007-0127996, filed Dec. 11, 2007, all of which are hereby
incorporated by reference herein in their entireties.
Claims
What is claimed is:
1. A method of treating an avian influenza viral infection in a
subject in need thereof, the method comprising administering an
effective amount of a composition comprising an extract of the bark
of Alnus japonica to the subject to treat the avian influenza viral
infection, wherein said extract of the bark of Alnus japonica is
prepared by a method comprising the steps of: (a) extracting the
bark of Korean indigenous Alnus japonica with 80.about.100% alcohol
at 30.about.80.degree. C.; and (b) recovering the resulting
extraction solution at the same temperature by vacuum concentrating
and drying.
2. The method of claim 1, wherein said alcohol is ethanol.
3. The method of claim 1, wherein the effective amount is between
about 0.01 and about 200 mg/kg subject per day.
4. The method of claim 1, wherein the effective amount is between
about 0.1 and about 100 mg/kg subject per day.
5. The method of claim 1, wherein the composition is administered
to the subject as a tablet.
6. The method of claim 1, wherein the avian influenza virus is
KBNP-0028 (KCTC 10866BP).
Description
TECHNICAL FIELD
The present invention relates to an antiviral composition
comprising an Alnus japonica extract, more specifically, relates to
a method for preparing an extract of the bark or stem of Alnus
japonica, which has high anti-influenza viral activity, and an
anti-influenza viral composition comprising the extract.
BACKGROUND ART
Avian influenza virus belongs to the orthomixoviridae family, and
causes damage to poultry such as chicken, turkey. Avian influenza
viruses are classified into 3 types of high-pathogenic,
low-pathogenic and non-pathogenic avian influenza viruses according
to the degree of pathogenicity, among which the high-pathogenic
virus is classified as an OIE List A disease by the World
Organization for Animal Health (OIE) and "a category 1 domestic
animal infectious disease" in Republic of Korea.
Influenza virus is classified as type A, B or C according to the
antigenicity of nucleocapsid protein and matrix protein. Moreover,
according to the difference of antigen structure of haemagglutinin
(HA) and neuraminidase (NA), the HA is classified into 16 subtypes
and NA is classified into 9 subtypes, wherein HA helps host cell
receptor binding, and fusion between host cell membrane and viral
envelope to cause virus infection and NA plays an important role
when virus buds out through the cell membrane after proliferation.
Theoretically, 144 kinds of virus subtypes could exist by the
combination of two proteins.
Infection generally occurs by contact with contaminated secretions,
furthermore, and it could be spread through the air, in both
particle and droplet forms, human feet, feed delivery vehicles,
apparatuses and feces on the surface of eggs etc. Although there
are various symptoms according to the pathogenicity of infecting
virus, generally, they are respiratory symptoms, diarrhea and a
sharp decline in egg production etc. Moreover, in some cases,
cyanosis appears in the head region such as crest, edema appears on
the face, or feathers are ruffled. Mortality rate also varies from
0% to 100% according to pathogenicity, but since the symptoms are
similar to those of Newcastle Disease, infectious
larynogotracheitis, mycoplasma infection and the like, an accurate
diagnosis is required.
High pathogenic avian influenza had occurred 23 times from 1959 to
2003 throughout the world, most of them were endemic and contained.
The outbreaks of highly pathogenic avian influenza subtype H5N1 had
occurred in Korea in December 2003, occurred in more than 30
countries including Europe, Africa and most countries in Southeast
Asia such as Japan, China, Thailand, Vietnam and Indonesia and thus
have become pandemic. Although it is known that humans cannot
become infected with avian influenza, prevention of avian influenza
is of paramount importance to public health sector since the case
of human infection with H5N1 in 1997, isolation of H9N2 avian
influenza viruses from humans in 1999 in Hong Kong and human cases
of H7 avian influenza infection in 2004 in Canada. According to a
report of the World Health Organization (WHO),
(http://www.who.int/csr/disease/avian_influenza/country/cases_table.sub.--
-2006.sub.--06.sub.--20/e n/index.html), it was confirmed that 228
persons had been infected with H5N1 subtype viruses and 130 persons
of them died during the period of 2003 to Jun. 20, 2006 in 10
countries. In Korea, since an outbreak of low pathogenic avian
influenza by H9N2 subtype viruses had occurred in 1996 and it
reoccurred in 1999.
If an avian influenza outbreak occurs, in most countries, the
poultry needs to be disposed of, and countries where avian
influenza outbreaks have occurred cannot export poultry products,
thus causing swingeing damages to poultry industry.
Furthermore, when there is a risk of human infection, the damages
spread to the whole industry including the tourism industry and the
transport industry, thus causing astronomical loss.
Natural substance refers to substances which are minimally
processed without artificial ingredients, and the natural
substances classified as GRAS (Generally Recognized As Safe) can be
used without restrictions on the quantity thereof or foods in which
the natural substances are to be used. In domestic industry, the
natural substances are classified as natural additives, and used as
food additives, and in foreign countries, it has been used as
health foods and medical supplies for user's purpose without extra
limitation, because of its excellent functionality.
Meanwhile, Alnus japonica is a deciduous, dicotyledonous tree in
the order Fagales, family Betulaceae, which is commonly called
Alnus japonica tree. They are distributed in Korea, Japan, China,
etc., and grow in marsh conditions, its height is about 20 m and
its bark is a deep purplish-brown color. Its winter bud is a long
oval shape just like the shape of an egg turned upside down, which
has three lines and a peduncle. The leaves of Alnus japonica grow
alternately, and they are oval shaped, egg-shaped (more or less
round on both ends, widest at the bottom) or lanceolate. Both sides
of a leaf are lustrous and leaf margins are saw-toothed. The
flowers of Alnus japonica bloom in March.about.April, are
unisexual, and form a catkin. Staminate spike bears staminate
flower and each bract subtends 3.about.4 flowers. There are four
perianths and four stamens in each flower. Fruit ripens in October
and 2.about.6 fruits are produced. It is long egg-shaped and looks
like a pine cone.
Examples of conventional patents relating to Alnus japonica
extracts include a cosmetic composition containing an Alnus
japonica extract (Korean Patent Publication No. 10-2003-0074500)
and a method for preparing a health drink useful for relieving
hangovers, which comprises extracts of Alnus japonica and green tea
leaves (Korean Patent Publication No. 10-2006-0023093), etc.
Recently, many research endeavors are taking place to develop
anti-viral agents throughout the world. Lamibudine used for the
treatment of HIV (Human Immunodeficiency Virus)-1 and hepatitis B,
gancyclovir used for the treatment of symptoms of herpes virus
infection, ribavirin which is used mainly for the treatment of
symptoms of respiratory syncytial virus infection but can be used
for the treatment of symptoms of various virus infection when it is
an emergency and zanamivir RELENZA.TM. and oseltamivir TAMIFLU.TM.
which are synthesized artificially as influenza virus neuraminidase
inhibitors are all commercially available after gaining approval.
However, use of amantadine and its analogue, rimantadine, which are
approved for treatment of influenza virus A, has decreased due to
the appearance of resistant virus and its side effect. Recently,
virus resistant to oseltamivir among H5N1 avian influenza viruses
appeared, therefore, there is an urgent need to develop various
antiviral agents.
The present inventors have confirmed antiviral activity of methanol
extract of Alnus japonica in Korean Patent Registration No.
10-0721703 and Korean Patent Registration No. 10-0769050. However,
the above mentioned patents have a disadvantage of showing
antiviral activity only when the extracts were administered at high
concentration and thus the possible applications thereof are
limited.
Therefore, the present inventors have made an extensive effort to
develop a natural substance having a low toxicity to normal cells,
while having an excellent effect of inhibiting influenza virus
proliferation even when administered at low concentration, and as a
result, confirmed that an extract obtained by extracting the bark
or stem of Alnus japonica, which is indigenous to Korea, with
8.about.90% ethanol at 30.about.80.degree. C. , has an excellent
anti-influenza virus effect, thereby completing the present
invention.
SUMMARY OF INVENTION
It is a main object of the present invention to provide a method
for preparing an extract of the bark or stem of Alnus japonica,
which has high anti-influenza viral activity.
It is another object of the present invention to provide a food
composition for preventing or improving influenza viral infection,
which comprises an extract of the bark or stem of Alnus japonica,
prepared by the above method.
It is still another object of the present invention to provide a
pharmaceutical composition for preventing or treating influenza
viral infection, which comprises an extract of the bark or stem of
Alnus japonica, prepared by the above method.
In order to achieve the above objects, the present invention
provides a method for preparing an extract of the bark or stem of
Alnus japonica, which has anti-influenza viral activity, the method
comprising the steps of: (a) extracting the bark or stem of Korean
indigenous Alnus japonica with 80.about.100% ethanol at
30.about.80.degree. C.; and (b) recovering the resulting extraction
solution.
The present invention also provides a food composition for
preventing or improving influenza viral infection, which comprises
an extract of the bark or stem of Alnus japonica, prepared by the
above method, and a sitologically acceptable supplemental
additive.
The present invention also provides a pharmaceutical composition
for preventing or treating influenza viral infection, which
comprises an extract of the bark or stem of Alnus japonica,
prepared by the above method, as an active ingredient.
The present invention also provides use of an extract of the bark
or stem of Alnus japonica prepared by the above method for
preventing or treating influenza viral infection.
The present invention also provides a method for preventing or
treating influenza viral infection using an extract of the bark or
stem of Alnus japonica prepared by the above method.
In the present invention, the influenza virus is preferably
selected from the group consisting of: human influenza virus, swine
influenza virus, equine influenza virus and avian influenza virus.
Preferably, the avian influenza virus is KBNP-0028 (KCTC
10866BP).
Other features and examples of the present invention will be
further clarified from the following detailed description and the
appended claims.
DETAILED DESCRIPTION OF THE INVENTION, AND PREFERRED
EMBODIMENTS
In one aspect, the present invention relates to a method for
preparing an extract of the bark or stem of Alnus japonica, which
has anti-influenza viral activity, the method comprising the steps
of: (a) extracting the bark or stem of Korean indigenous Alnus
japonica with 80.about.100% ethanol at 30.about.80.degree. C.; and
(b) recovering the resulting extraction solution.
In one embodiment of the present invention, after the bark or stem
of Alnus japonica was powdered and extracted with water or ethanol
by hot water extraction, cold water extraction, reflux-cooling
extraction or ultrasonic extraction, then centrifuged, thus
obtaining an extract of the bark or stem of Alnus japonica.
In the present invention, after a composition containing an extract
of the bark or stem of Alnus japonica was added to SPF embryonated
eggs infected with avian influenza virus and cultured, the plate
hemagglutination test was performed, and as a result, it was
confirmed that the composition containing an extract of the bark or
stem of Alnus japonica has excellent anti-influenza viral effect
even when administered at low concentration.
In another aspect, the present invention relates to a
pharmaceutical composition for preventing or treating influenza
viral infection (influenza viral disease), which comprises an
extract of the bark or stem of Alnus japonica, prepared by the
above method, as an active ingredient.
In the present invention, influenza virus is preferably selected
from the group consisting of: human influenza virus, swine
influenza virus, equine influenza virus, and avian influenza virus.
More preferably, avian influenza virus is KBNP-0028 (KCTC
10866BP).
The inventive extract of the bark or stem of Alnus japonica is a
natural substance and thus has no toxicity, which enables long-term
administration in high dosage as a medical product.
A composition comprising the inventive extract of the bark or stem
of Alnus japonica can be prepared by mixing together with
pharmaceutical agents such as antihistaminic agents,
anti-inflammatory analgesic agents, anti-cancer agents and
antibiotics, or can be used in combination therewith.
The composition of the present invention in pharmaceutical dosage
forms may be used in the form of pharmaceutically acceptable salts,
and also may be used alone or in appropriate association, as well
as in combination with other pharmaceutically active compounds.
The pharmaceutical composition comprising the inventive extract may
be formulated into an oral preparation such as powders, granules,
tablets, capsules, suspensions, emulsions, syrups, aerosols etc, an
external preparation, a suppository and a sterile injectable
solution, according to the conventional preparation methods. The
pharmaceutical composition comprising the inventive extract may
comprise carriers, excipients and diluents, and examples of
suitable carriers, excipients and diluents include lactose,
dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol,
maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate,
calcium silicate, cellulose, methyl cellulose, microcrystalline
cellulose, polyvinyl pyrrolidone, water, methylhydroxy benzoate,
propylhydroxy benzoate, talc, magnesium stearate and mineral
oil.
The composition of the present invention can be formulated into a
preparation form, together with the conventional diluents or
excipients such as fillers, extenders, binders, wetting agents,
disintegrants, surfactants etc. A solid preparation for oral
administration includes tablets, pills, powders, granules, capsules
etc, and the solid preparation is formulated by mixing the extract
with at least one excipient, for example, starch, calcium
carbonate, sucrose, lactose, gelatin and the like. Also, a
lubricant such as magnesium stearate and talc is used in addition
to the excipients. A liquid preparation for oral administration
includes suspension, zipeprol, emulsion, syrup and the like, and
various excipients, for example, wetting agents, flavoring agents,
fragrances, preservatives etc, can be contained thereto in addition
to conventional diluents such as water and liquid paraffin A
preparation for parenteral administration includes sterile aqueous
solution, non-aqueous solution, suspensions, emulsions, a
lyophilized preparation, and suppositories. Examples of non-aqueous
solution and suspensions include vegetable oil such as propylene
glycol, polyethylene glycol and olive oil, and injectable esters
such as ethyloleate and the like. Base materials of suppositories
include witepsol, macrogol, tween 60, cacao butter, laurin butter,
glycerol gelatine and the like.
Although the dosage of the extract according to the present
invention varies depending on the weight and condition of a
patient, the severity of the disease, the dosage form,
administration route, and treatment period, the dosage can be
properly determined by a person skilled in the art. However, in
order to achieve the desired effect, the inventive extract is
administered at a dose of 0.01-200 mg/kg per day, preferably
0.1-100 mg/kg per day. The foregoing doses may be administered as a
single dose or may be divided into multiple doses per day, and the
doses do not limit the scope of the present invention in any
way.
The inventive extract can be administered to mammals including
rats, mice, domestic animals, humans and the like via various
routes. The mode of administration may include, for example, oral
and rectal administration, or venous, muscular, subcutaneous,
endometrium or intracerebroventricular injections.
In another aspect, the present invention provides a food
composition for preventing or improving influenza viral infection,
which comprises an extract of the bark or stem of Alnus japonica,
prepared by the above method, and a sitologically acceptable
supplemental additive.
In an embodiment of this aspect, the influenza virus is preferably
selected from the group consisting of: human influenza virus, swine
influenza virus, equine influenza virus and avian influenza virus.
More preferably, the avian influenza virus is KBNP-0028 (KCTC
10866BP).
The composition comprising the inventive extract or
pharmaceutically acceptable salts thereof can be used as a main
ingredient, food additive and supplement when preparing various
functional foods and health functional foods.
In the present invention, the term "functional foods" refers to a
food whose functionality is improved by adding the inventive
extract thereto. Functionality can be broadly divided into physical
functionality such as synthetic flavors and natural flavors
physiological functionality. When the inventive extract is added to
a general food, such as synthetic flavors and natural flavors and
physiological functionality thereof will be improved. Therefore, in
the present invention, the food with improved functionality is
broadly defined as functional foods.
Aside from the above, the inventive extract may contain various
nutrients, vitamins, minerals (electrolytes), flavors such as
synthetic flavors and natural flavors, coloring matters, enhancer
(cheese, chocolate, etc.) pectic acid and its salts, alginic acid
and its salts, organic acids, protective colloid thickners, pH
control agents, stabilizers, preservatives, glycerins, alcohols and
carbonating agents for carbonated beverage use, etc. In addition,
the extract of the present invention may contain natural fruit
juices, and fruit pulps for the provision of fruit juice drinks and
vegetable drinks These ingredients can be used independently or in
combination. The proportion of these additives is not so critical,
but generally selected from the range of 0.01.about.20 parts by
weight based on 100 parts by weight of the inventive extract.
EXAMPLES
Hereinafter, the present invention will be described in more detail
by examples. However, it is obvious to a person skilled in the art
that these examples are for illustrative purpose only and are not
construed to limit the scope of the present invention.
Example 1
Preparation of Alnus japonica Extract
1-1: Preparation of Extraction Solvent and Extraction at Various
Temperatures
The bark of Korean indigenous Alnus japonica purchased from
Kyungdong market, Seoul, Korea was dried at room temperature for 24
hrs, finely chopped and pulverized. 1 kg of the obtained bark
fragments were extracted with 10L of 95% ethanol under reflux for 8
hrs at 40.degree. C., 60.degree. C. and 80.degree. C. or extracted
with 10L water under reflux for 4 hrs at 100.degree. C., and
filtered under vacuum to collect supernatant, followed by eluting
useful substances from the obtained fragments. The eluted useful
substances are dried under vacuum for 24 hrs to obtain 100 g of
Alnus japonica powder, and the obtained powder was dissolved in
99.9% dimethyl sulfoxide (DMSO) solution to a concentration of 20
mg/ml, then used for the following experiments.
For comparison experiments, Alnus japonica bark extracts prepared
by the method in example 1 of Korean Patent Registration No.
10-0721703 and Korean Patent Registration No. 10-0769050 (an
antiviral composition comprising Alnus japonica extracts), as a
control group.
1-2: Preparation of Alnus japonica Extracts using Alnus japonica
from Different Origins
The bark and stem of Korean indigenous Alnus japonica purchased
from Kyungdong market, Seoul, Korea and the bark of Chinese native
Alnus japonica purchased from Yanbian market in China were dried at
room temperature for 24 hrs, finely chopped and pulverized. 1 kg of
the obtained Alnus japonica fragments were extracted with 10L of
80% ethanol and 95% ethanol under reflux at 40.degree. C.,
respectively, and filtered under vacuum to collect supernatant,
followed by eluting useful substances from the obtained fragments.
The eluted useful substances are dried under vacuum for 24 hrs to
obtain 100 g of Alnus japonica powder was obtained, and the
obtained powder was dissolved in 99.9% dimethyl sulfoxide (DMSO)
solution to a concentration of 20 mg/ml, then used for the
following experiments.
Example 2
Examination of Anti-Viral Effect of Alnus japonica Extracts
2-1: Preparation of KBNP-0028
As avian influenza virus used in the experiment, hyperproliferative
KBNP-0028 (KR 2006-0026591) cloned after subculturing
A/chicken/Korea/SNU0028/2000(H9N2) virus (it is isolated in Korea
in 2000) in chick embryo was used. That is, SNU0028
[A/chicken/Korea/SNU0028/2000(H9N2); isolation and report to
National Veterinary Research and Quarantine Service, May 9, 2005]
is low-pathogenic avian influenza virus of H9N2 subtype, and
isolated from chickens showing mortality and egg drop syndrome in a
chicken farm located in North jeola Province in Jan. 28, 2000. The
isolation method is as follows: after kidney and tracheal samples
from infected chickens are dissolved, suspended in phosphate
buffer, and filterated with 0.45 .mu.m filter paper, each sample is
inoculated into three allantoic cavities of SPF (Specific Pathogen
Free) embryonated egg (Sunrise Co., NY), and cultured at 37.degree.
C. to obtain allantoic fluid. 20 .mu.l of the allantoic fluid and
20 .mu.l of 0.1% chicken red blood cells, extracted from a chicken
hatched from the SPF embryonated egg, are dropped on a glass plate,
and mixed to carry out the plate hemagglutination test.
As a result, in all of the allantoic fluids, obtained by
inoculating the kidney sample and tracheal sample, hemagglutination
occurred. The virus was identified with RT-PCR using H9N2 specific
primer and base sequence analysis (Kim Min Chul, Master's Thesis,
2002, Seoul National University), and stored at -70.degree. C.
Among them, the virus isolated from tracheal sample was used in the
experiment.
In order to select a vaccinia strain having high productivity in
embryonated eggs, the SNU0028 was diluted with a phosphate buffer
solution to a concentration of 0.05 to 0.5 HAU/ml, and 200 .mu.l of
the diluted solution was inoculated into the allantoic cavity of
10-11-day-old SPF embryonated eggs (Sunrise Co., NY), then cultured
for three days at 37.degree. C. Every day, the embryonated eggs,
which died three days ago, were discarded through egg examination
in the morning and afternoon. The embryonated eggs, which survived
for three days, were stored for 12.about.24 hrs at 4.degree. C.,
from which allantoic fluid was harvested to measure the volume and
hemagglutination titer of each egg. Among them, allantoic fluid
having the most quantity and the highest hemagglutination titer was
inoculated into embryonated eggs using the same method as described
above, and subcultured 19 times to select allantoic fluid whose
productivity was increased showing high hemagglutination titer and
high yield thereof, and thus, the strain was named KBNP-0028 and
deposited in GenBank located Eoeundong, Youseonggu, Daejeon city,
Korea on Oct. 26, 2005 (KCTC 10866BP).
2-2: Culturing Embryonated Egg Shell Fragments
The egg shell of 10.about.11-day-old SPF embryonated eggs (Sunrise
Co., NY) was washed with 70% ethanol and chick embryo and all body
fluids were removed. The resulting egg shell was cut into pieces
about 8 mm long and 8 mm wide while maintaining villi and allantois
adhered to the inner surface of the egg shell, and each piece was
added into a 24-well culture plate. Culture medium was prepared by
(i) mixing 199 medium (GIBCO-BRL, NY, USA) with F10 medium
(GIBCO-BRL, NY, USA) at a ratio of 1:1, (ii) adding 0.075% of
sodium bicarbonate and 100 .mu.g/ml of gentamicin.
The 10.about.11-day-old SPF embryonated eggs (Sunrise Co., NY) were
infected with virus by adding 100 .mu.l of the crude allantoic
fluids, KBNP-0028 prepared in Example 2-1, which is 4.about.10-fold
diluted, to the villi and allantois of embryonated egg shell
fragments, and culturing for 30 min at 37.degree. C., and added
with 1000 .mu.l of the culture medium, then Alnus japonica extracts
prepared in Example 1-1 and Example 1-2 was added to 6 well plates
at various concentration, respectively, followed by culturing for 7
days at 37.degree. C.
2-3: Test of Antiviral Effect
Culture broth of said virus-infected fluids cultured for 7 days in
Example 2-2, which is added with Alnus japonica extracts at various
concentrations, was taken to carry out plate hemagglutination test.
25 .mu.l of the culture broth and 25 .mu.l of chicken red blood
cells (0.1%) were dropped on a glass plate and mixed evenly. Virus
proliferation was determined according to whether hemagglutination
occurred within 2 min by moving the glass plate right and left, and
up and down.
TABLE-US-00001 TABLE 1 Control Alnus japonica extracts (.mu.g/ml)
virus non-virus Extract solvent 400 200 100 50 6/6 0/6 80.degree.
C., 95% ethanol 0/6 0/6 2/6 5/6 60.degree. C., 95% ethanol 0/6 0/6
2/6 5/6 40.degree. C., 95% ethanol 0/6 0/6 1/6 3/6 100.degree. C.,
water 1/6 4/6 6/6 6/6 99.9% methanol 0/6 2/6 6/6 --
As a result, as shown in Table 1, in the sample added with
100.degree. C. water extract as a negative control,
hemagglutination occurred in one of 6 test samples at 400 .mu.g/ml,
showing partial antiviral effect, however, hemagglutination
activity was shown in all of 6 test samples at 50 .mu.g/ml and 100
.mu.g/ml, suggesting that virus proliferation was not
inhibited.
On the other hand, in the sample added with the 95% ethanol extract
(at 80.degree. C.), hemagglutination did not occur in all of 6 test
samples at 400 .mu.g/ml and 200 .mu.g/ml showing that virus
proliferation was completely inhibited, hemagglutination occurred
in two of 6 test samples at 100 .mu.g/ml, showing partial antiviral
effect, and hemagglutination occurred in five of 6 test samples at
50 .mu.g/ml, showing weak antiviral effect. The sample added with
95% ethanol extract (at 60.degree. C.) showed the same
hemagglutination results as those of the 95% ethanol extract (at
80.degree. C.). The 95% ethanol extract (at 40.degree. C.) showed
the same hemagglutination as those of the 95% ethanol extracts (at
80.degree. C., 65.degree. C., respectively) at 400 .mu.g/ml and 200
.mu.g/ml, and thus, no hemagglutination activity was shown in all
samples, suggesting that viral proliferation was completely
inhibited, and showed partial antiviral effect hemagglutination
occurred in one of 6 test samples at 100 .mu.g/ml and three of 6
test samples at 50 .mu.g/ml, thus confirming that the extract shows
high antiviral at various effect even at low concentration.
Based on extract concentration of 100 .mu.g/ml, antiviral activity
at various solvents was in the order: 95% ethanol extract (at
40.degree. C.)>95% ethanol extract (at 80.degree. C.) and 95%
ethanol extract (at 60.degree. C.)>water extract (at 100.degree.
C.). Therefore, it was determined that 95% Alnus japonica extract
(at 40.degree. C.) is most suitable as the Alnus japonica extract
to prepare the inventive antiviral composition.
In order to compare antiviral effects according to Alnus japonica
with different origins, extracted at 40.degree. C. at which the
highest inhibition effect on virus proliferation was shown and
various extract solvents, the same experiment as described above
was performed under conditions shown in Table 2.
TABLE-US-00002 TABLE 2 Control Alnus japonica non- Raw materials to
be extracts (.mu.g/ml) virus virus extracted and solvents 100 50 25
12.5 6/6 0/6 the bark of Korean native 2/6 4/6 6/6 6/6 Alnus
japonica 80% ethanol extract the bark of Korean native 1/6 3/6 4/6
5/6 Alnus japonica 95% ethanol extract the stem of Korean native
3/6 5/6 6/6 6/6 Alnus japonica 80% ethanol extract the stem of
Korean native 1/6 3/6 6/6 6/6 Alnus japonica 95% ethanol extract
the bark of Chinese native 1/6 4/6 6/6 6/6 Alnus japonica 80%
ethanol extract the bark of Chinese native 1/6 5/6 6/6 6/6 Alnus
japonica 95% ethanol extract
As a result, as shown in Table 2, it was observed that 80% and 95%
ethanol extracts of the bark of Korean indigenous Alnus japonica
showed excellent antiviral activity, compared to 80% and 95%
ethanol extracts of the stem of Korean indigenous Alnus japonica.
Thus, it could be confirmed that the bark was suitable for use as
Alnus japonica extract for the inventive antiviral composition.
When comparing Alnus japonica from different origins, 80% ethanol
extracts of the bark of Korean indigenous Alnus japonica and
Chinese indigenous Alnus japonica showed similar activity, and 95%
ethanol extract of the bark of Korean native Alnus japonica showed
more excellent activity than that of 95% ethanol extract of Chinese
native Alnus japonica, and when comparing antiviral activities at
various extract solvents, 95% ethanol extract showed higher
antiviral activities than that of 80% ethanol extract.
From the above the results, it could be confirmed that 95% ethanol
extract of the bark of Korean native Alnus japonica extracted at
40.degree. C. shows the most excellent antiviral effect.
Hereinafter, examples of preparations of the pharmaceutical
composition comprising an Alnus japonica extract according to the
present invention, however, these examples are for illustrative
purpose only and are not construed to limit the scope of the
present invention.
Preparation Example 1
Powder Preparation
Extract of Alnus japonica: 20 mg
Lactose: 100 mg
Talc: 10 mg
The above ingredients were mixed, and changed in an air-tight pack
to prepare a powder preparation
Preparation Example 2
Tablet Preparation
Extract of Alnus japonica: 10 mg
Cornstarch: 100 mg
Lactose: 100 mg
Stearin magnesium: 2 mg
The above ingredients were mixed, and tableted according to the
conventional method to prepare tablets.
Preparation Example 3
Capsule Preparation
Extract of Alnus japonica: 20 mg
Crystalline cellulose: 13.3 mg
Lactose: 65.8 mg
Magnesium stearate: 0.9 mg
The above ingredients were mixed, and changed in a gelatin capsule
according to the conventional method to prepare capsules.
Preparation Example 4
Injection Preparation
Extract of Alnus japonica: 10 mg
Mannitol: 180 mg
Sterile distilled water for injection: 2,974 mg
Na.sub.2HPO.sub.4.12H.sub.2O: 26 mg
The above ingredients were added to an ample at the amount shown
above per ampule (3 ml) according to the conventional preparation
method of injectable solution.
Preparation Example 5
Liquid Preparation
Extract of Alnus japonica: 20 mg
Isomerized sugar: 10 g
Mannitol: 5 g
Proper quantity of purified water
Each ingredient was dissolved in the purified water and added with
a suitable amount of lemon flavor to mix the above ingredients,
then the purified water was added to a total volume of 100 ml,
followed by sterilizing to change in a brown vial, thus preparing
liquid preparation, according to the conventional liquid
preparation method.
INDUSTRIAL APPLICABILITY
As described above in detail, the extract of the bark or stem of
Alnus japonica according to the present invention has low toxicity
to choriollantonic cells which is a normal cell, while having an
excellent antiviral effect even when administered at low
concentration. Therefore, the composition comprising the Alnus
japonica extract can be used effectively in preventing and treating
influenza viral infection.
Although the present invention has been described in detail with
reference to the specific features, it will be apparent to those
skilled in the art that this description is only for a preferred
embodiment and does not limit the scope of the present invention.
Thus, the substantial scope of the present invention will be
defined by the appended claims and equivalents thereof. It is
understood that numerous changes and modifications can be made by
those skilled in art without departing from the invention concepts
disclosed herein.
* * * * *
References